J. Nannoplankton Res. 32 (1), 2011, pp29-34 © 2011 International Nannoplankton Association
ISSN 1210-8049 Printed by Harcourt Colour Print, Swansea, UK

29

The morphological response of Emiliania huxleyi toseawater carbonate chemistry changes: an inter-strain
comparison
Gerald Langer

ICTA, Autonomous University of Barcelona (UAB), 08193 Bellaterra, Spain: Gerald.Langer@awi.de

Ian Probert

CNRS / UPMC, Station Biologique de Roscoff, 29682 Roscoff, France

Gernot Nehrke

Alfred Wegener Institute for Polar and Marine Research, 27570 Bremerhaven, Germany

Patrizia Ziveri

FALW, Vrije Universiteit Amsterdam, 1081HV Amsterdam, The Netherlands
Manuscript received 19th June, 2010; revised manuscript accepted 22nd November, 2010.

Abstract Four strains of the coccolithophore Emiliania huxleyi (RCC1212, RCC1216, RCC1238, RCC1256)
were grown in dilute batch culture at four CO2 levels ranging from ~200 µatm to ~1200 µatm. Coccolith morphology
was analyzed based on scanning electron micrographs. Three of the four strains did not exhibit a change in morphology over the CO2 range tested. One strain (RCC1256) displayed an increase in the percentage of malformed coccoliths
with increasing CO2 concentration. We conclude that the sensitivity of the coccolith-shaping machinery to carbonate
chemistry changes is strain-specific. Although it has been shown before that carbonate chemistry related changes ingrowth- and calcification rate are strain-specific, there seems to be no consistent correlation between coccolith morphology and growth or calcification rate. We did not observe an increase in the percentage of incomplete coccoliths
in RCC1256, indicating that the coccolith-shaping machinery per se is affected by acidification and not the signalling
pathway that produces the stop-signal for coccolith growth.
Keywords Emiliania huxleyi, morphology, carbonate chemistry
1. Introduction
In the context of ocean acidification, i.e. the decreaseof surface seawater pH due to anthropogenic CO2 emissions (Royal Society, 2005), the response of marine
calcifiers to altered seawater carbonate chemistry hasreceived particular attention. Coccolithophores, unicellular haptophyte algae that cover the cell surface withintracellularly-produced calcite platelets (coccoliths),
have been shown to change growth rate and calcite- andorganic carbon production in response to seawater carbonate chemistry changes (Riebesell et al., 2000; Langer
et al., 2006; Feng et al., 2008). It is generally assumed
that a decrease in calcite production, the most commonly
observed response to seawater acidification, is detrimental for a coccolithophore. However, the function of calcification is unknown (Young, 1994) and so far there isno evidence that calcification plays a physiological role
(Trimborn et al., 2007). Nevertheless, the products ofcalcification, i.e. the coccoliths and the resulting coccosphere, are most likely advantageous for the organism. In an Atomic Force Microscopy study of coccoliths,
the authors concluded that “clearly, millions of years ofnatural selection have perfected the tailoring of coccolith biocrystals so that the mineral structure of the material is used to the greatest advantage” (Henriksen et al.,2003). To better understand the potential consequencesof ocean acidification for coccolithophores, it is therefore important to study the effect of changing seawater carbonate chemistry not only on calcite production
but also on coccolith morphology. The latter has beenshown to be influenced by salinity (Green et al., 1998;

Bollmann and Herrle, 2007) and temperature (Watabe
and Wilbur, 1966; Langer et al., 2010). The carbonate
chemistry effect on morphology was shown to be species specific (Langer et al., 2006). Moreover, conflicting
results have been published on the response of a single
species, namely Emiliania huxleyi. In the seminal study
by Riebesell et al. (2000) an increase of the percentage
of malformed coccoliths with increasing CO2 concentration was reported. In contrast, no differences in coccolith morphology were found in a later study (Feng et al.,2008). However, coccolith morphology was not quantified in either of these studies, and in all of the studies
cited above only one culture strain of E. huxleyi (and inevery study a different strain) was used. Since the effectof carbonate chemistry changes on growth rate and calcite production in E. huxleyi is strain specific (Langer etal., 2009), it is probable that the effect on coccolith morphology is likewise. In order to test this hypothesis, weanalyzed the coccolith morphology of the four E. huxleyi
strains used by Langer et al. (2009).
2. Material and Methods
Clonal cultures of Emiliania huxleyi (strains RCC1212,
RCC1216, RCC1238, and RCC1256) were grown in aged,
sterile-filtered (0.2µm pore-size cellulose-acetate filters)
North Sea seawater enriched with 100 µmol L-1 nitrate,
6.25 µmol L-1 phosphate, and trace metals and vitamins
as in f/2 medium (Guillard and Ryther, 1962). The strains
were obtained from the Roscoff Culture Collection (www.
sb-roscoff.fr/Phyto/RCC). Cultures were grown under a
16/8 hour light/dark cycle. Experiments were carried out

30Langer, Probert, Nehrke, Ziveri

at a light intensity of 400 µmol photons m-2s-1 in an adjustable incubator (Rubarth Apparate GmbH, Germany). The
temperature used was 17°C for RCC1216 and RCC1256,
and 20°C for RCC1212 and RCC1238. We chose different temperatures as opposed to a standard temperature inorder to grow each strain near its optimum temperaturefor growth. Cells were pre-adapted to experimental conditions for approximately 12 generations and grown in dilute batch cultures (Langer et al., 2009). Each data point
presented in the tables and figures is the mean value of
triplicate culture experiments. CO2 levels were adjusted
by adding calculated amounts of HCl or NaOH to the medium. For further experimental details see Langer et al.
(2009).
Samples for scanning electron microscope analysis
were filtered onto polycarbonate filters (0.8 μm pore size),dried in a drying cabinet at 60°C for 24 hours, then sputter-coated with gold-palladium. Imaging was performed
with a Philips XL-30 digital scanning field-emission elec-tron microscope. Four categories were used to describe
the morphology of Emiliania huxleyi: ‘normal’, ‘malformed’, ‘incomplete’, and ‘incomplete and malformed’coccoliths (for reference images for the categories, see
Figures 1-4). An average of approximately 350 coccoliths
was analyzed per sample (Langer and Benner, 2009).
3. Results and Discussion

3.1. Response patterns
Strain RCC1238 displayed more than 90% normallygrown coccoliths under all CO2 concentrations tested(Figure 5, Table 1). The same held for strain RCC1216(Figure 6, Table 1). Both RCC1238 and RCC1216 aretherefore considered as insensitive over the CO2 ranges
tested. We draw the same conclusion for strain RCC1212(Figure 7, Table 1), although the result was less obvious.
In RCC1212 a trend was observed towards a higher percentage of normal coccoliths under higher CO2 concentration. This has to be inexperimentCO2
normalmalformed
incomplete malformed +terpreted with caution,[µatm]
incomplete
however, because thedifferences were relaRCC1238 1
206
94.8
2.2
2.4
0.6
tively small and the stanSD
2.1
0.9
1.0
0.8
dard deviation relatively
RCC1238 2
395
93.9
1.7
3.3
1.1
high (Table 1). The deSD
0.7
0.4
1.3
0.8
crease in the percentage
RCC1238 3
681
94.8
1.4
2.5
1.4
SD
2.0
0.6
0.9
0.6
of normal coccoliths
RCC1238 4
929
93.3
0.5
2.8
3.4
with increasing CO2 in
SD
1.5
0.5
1.1
1.0
RCC1256 (Figure 8,
Table 1) is, however,
RCC1216 1
218
96.8
1.4
1.6
0.2
meaningful because the
SD
1.7
0.7
1.1
0.3
standard deviation was
RCC1216 2
422
97.6
0.6
1.5
0.2
much smaller and the
SD
0.1
0.2
0.3
0.2
difference in question,
RCC1216 3
729
97.8
0.6
1.5
0.1
i.e. the morphology at
SD
0.1
0.4
0.2
0.2
the highest CO2 level
RCC1216 4
1201
92.2
3.1
1.8
2.9
compared to the other
SD
4.2
1.5
1.0
1.7
levels, was considerablylarger. Hence, coccolith
RCC1212 1
194
51.0
42.8
4.0
2.3
morphogenesis of this
SD
5.0
5.2
0.8
2.3
strain was impaired by
RCC1212 2
409
58.5
35.7
4.0
1.8
acidification of seawater.
SD
2.9
1.6
1.9
0.5
The absolute level
RCC1212 3
752
66.0
27.5
4.0
2.6
of aberrant coccoliths
SD
3.5
4.3
0.6
0.3
in RCC1212 was much
RCC1212 4
1096
65.2
28.2
2.2
4.4
higher than in the other
SD
5.6
4.4
0.5
1.6
strains (Table 1). The
reason for this is unRCC1256 1
193
97.0
1.2
1.5
0.2
known. In general, the
SD
1.1
0.2
0.9
0.4
base level of malforRCC1256 2
399
97.3
1.0
1.6
0.2
mation in cultured cocSD
0.6
0.4
0.7
0.2
colithophores is highly
RCC1256 3
587
91.3
7.0
0.7
1.0
variable (Langer and
SD
0.8
1.2
0.2
0.3
Benner, 2009), and as a
RCC1256 4
915
61.0
29.4
1.4
8.2
rule cultured specimens
SD
2.5
1.5
1.1
1.8
display a higher percentage of malformed cocTable 1 Coccolith morphology (percentage of counted coccoliths) at different CO2 levels. CO2 partial pressurescoliths than specimens

were taken from Langer et al. (2009). SD = standard deviation.

Carbonate chemistry and coccolith morphology

31

Figure 1 SEM images of Emiliania huxleyi (RCC1238) coccoliths. A)
Normal B) Malformed C) Incomplete D) Incomplete and malformed.
All coccoliths in distal view. All scalebars 2 µm.

Figure 2 SEM images of Emiliania huxleyi (RCC1216) coccoliths. A)
Normal B) Malformed C) Incomplete D) Incomplete and malformed.
All coccoliths in distal view. All scalebars 2 µm.

Figure 3 SEM images of Emiliania huxleyi (RCC1212) coccoliths. A)
Normal B) Malformed C) Incomplete D) Incomplete and malformed.
All coccoliths in distal view. All scalebars 2 µm.

Figure 4 SEM images of Emiliania huxleyi (RCC1256) coccoliths. A)
Normal B) Malformed C) Incomplete D) Incomplete and malformed.
All coccoliths in distal view. All scalebars 2 µm.

from oceanic samples (Langer et al., 2006). The cause of
the latter observation, often termed “culture artefacts”,
is also unknown (Langer and Benner, 2009). However,for the interpretation of our results it is important to notethat the absolute level of malformation has been shown tohave no influence on the response to carbonate chemistrychanges in Calcidiscus leptoporus (Langer et al., 2006).
Given that this also holds for E. huxleyi, the response of
RCC1212 can be compared to the responses of the otherstrains.
It is therefore concluded that the four strains tested
displayed two types of responses: either no change inmorphology (RCC1238, RCC1216, and RCC1212), or ahigher percentage of malformed coccoliths at a CO2 partial pressure of ca. 900 µatm (RCC1256).

3.2. Strain specific responses
Since the initial study of Riebesell et al. (2000), the latter
response pattern (i.e. higher malformation at higher CO2)
has been regarded as typical for E. huxleyi and indeed forcoccolithophores. In our study, however, this response appears to be the exception rather than the rule. The originof the differences in response remains to be discovered.
Several environmental factors, which could presumably
influence the response pattern, were discussed elsewhere(Langer et al., 2009). Distance from the shore of the siteof strain isolation and morphotype of the strain can beruled out (Langer et al., 2009). For details on the sites ofstrain isolation see also Table 2 and Figure 9. It may seemlogical to assume that surface seawater CO2 concentration
at the site of strain isolation is a main factor influencing

32Langer, Probert, Nehrke, Ziveri

Figure 5 Percentage of normal (n), malformed (m), incomplete (i), malformed and incomplete (m/i) coccoliths (strain RCC1238) vs. CO2 partial pressure. Values represent an average of triplicate experiments. CO2
partial pressures were taken from Langer et al., (2009).

Figure 6 Percentage of normal (n), malformed (m), incomplete (i), malformed and incomplete (m/i) coccoliths (strain RCC1216) vs. CO2 partial pressure. Values represent an average of triplicate experiments. CO2
partial pressures were taken from (Langer et al., 2009).

Figure 7 Percentage of normal (n), malformed (m), incomplete (i), malformed and incomplete (m/i) coccoliths (strain RCC1212) vs. CO2 partial pressure. Values represent an average of triplicate experiments. CO2
partial pressures were taken from (Langer et al., 2009).

Figure 8 Percentage of normal (n), malformed (m), incomplete (i), malformed and incomplete (m/i) coccoliths (strain RCC1256) vs. CO2 partial pressure. Values represent an average of triplicate experiments. CO2
partial pressures were taken from (Langer et al., 2009).

the response pattern, see also Langer et al. (2009). Southof Iceland, where RCC1256 was isolated, the surface water CO2 partial pressure ranges from ca. 270 µatm in August to ca. 360 µatm in February (Takahashi et al., 2002).
The CO2 partial pressure at the three other sites of isolation is comparatively constant 320 µatm, Takahashi et al.,(2002). However, it appears unlikely that the strain thathas historically experienced varying CO2 levels over thecourse of the year is particularly sensitive to acidification,
whereas strains that have experienced quasi-constant carbonate chemistry are insensitive. This notion is strengthened when taking into account not only morphology but
also growth rate, calcite- and organic carbon production(Langer et al., 2009). At any rate, all four strains may be
regarded as belonging to different populations with specific genetic features (Langer et al., 2009), rendering it
reasonable to attribute the different response patterns tothese genetic features.

3.3. Comparison with literature data
Considering all available data on coccolithophores, there
seems to be no consistent correlation between coccolithmorphology and growth or calcification rate in carbonate chemistry manipulation experiments. In RCC1212,
RCC1216 (Table 1, Langer et al., 2009), and CCMP371(Feng et al., 2008) changes in growth and calcification rate,
but no change in morphology, were observed. In RCC1256(Table 1, Langer et al., 2009) changes in growth and calcification rate and morphology were observed, whereas
B92/11 (Riebesell et al., 2000) and RCC1135 (Langer etal., 2006) changed calcification rate and morphology, but
not growth rate. Finally, RCC1238 (Table 1, Langer et al.,2009) and RCC1200 (Langer et al., 2006) did not show achange in any of these parameters. These response patterns
are difficult to interpret because the adverse effects of seawater acidification on coccolithophore calcification are notunderstood on a process level. It is not even known which

Carbonate chemistry and coccolith morphology

33

Figure 9 Map showing the sites of strain isolation. Map modified from http://www.justmaps.org/maps/thematics/physical.asp
Strain CodeIsolation
Date

Location of sample
from which culture
isolated

SeawaterMax. annual SST
temperature at
at samplingtime of sampling location

RCC12129/2000

RCC1216

9/1998

RCC1238

11/2005

RCC1256

7/199934°28’S 17°18’E 15°C
(South Atlantic,
off South Africa)
42°18’S 169°50’E 11°C
(Tasman Sea,
off New Zealand)
34°01’N 139°50’ E 18°C
(North Pacific,
off Japan)
63°24’N 20°20W 9°C(North Atlantic,
off Iceland)Experimental
temperature

Morphotype

Isolated by

21°C

20°C

B

Ian Probert

18°C

17°C

R

Ian Probert

25°C

20°C

A

Ian Probert

14°C

17°C

A

Ian Probert

Table 2 Information on culture strains used in this study. Max. annual SST was taken from the World Ocean Atlas (www.nodc.noaa.gov).

parameter of the carbonate system causes these effects.
It was hypothesised that seawater pH changes upset
the otherwise strictly controlled ion balance at the plasma
membrane and thereby disturb intracellular ionic composition (Langer et al., 2006). This, in turn, would disrupt
the specific function of polyelectrolytes involved in coccolithogenesis. Two types of polyelectrolytes have beenshown to be involved in coccolithogenesis, namely coccolith-associated polysaccharides (Fichtinger-Schepman
et al., 1981; Borman et al., 1982; Marsh et al., 2002; Henriksen et al., 2004; Henriksen and Stipp, 2009) and twoelements of the cytoskeleton (Langer et al., 2010). In thelatter study it was also shown that a malfunction of thecytoskeleton leads to a decreased growth rate as well as anincreased percentage of malformed coccoliths. The lack ofa correlation between morphology and growth rate in carbonate chemistry manipulation experiments (see above)
suggests that the cytoskeleton is not the polyelectrolyte

which is affected by acidification. It may, therefore, beworthwhile focusing research regarding this question on
coccolith-associated polysaccharides.
It is also noteworthy that the percentage of incomplete coccoliths did not change in any of the tested strains(Table 1). In the context of developing a process-based
understanding it is significant that the increase in malformations in higher than ambient CO2 concentrations in
RCC1256 (Table 1) was not accompanied by an increase
in incomplete coccoliths, matching the observation for C.
leptoporus RCC1135 (Langer et al., 2006). These observations clearly suggest that when acidification does havean effect, it is on morphogenesis per se and not the signalling pathway that produces the stop-signal for coccolith
growth. This conclusion renders the overall lack of correlation between coccolith morphology and cellular calcite
content / calcification rate (see above) plausible.

34

Langer, Probert, Nehrke, Ziveri

4. Conclusion
On the whole, the data presented here show firstly that themorphological response of E. huxleyi to short term acidification of seawater does not always correlate with the calcification or growth rate response. This finding is particularly
relevant if it should indeed turn out that the product of calcification, i.e. the coccoliths, and not the process of calcification itself is of key importance for a coccolithophore.
Secondly, it is shown that the morphological response isstrain specific, once more highlighting the need to consider
the potential for adaptation of coccolithophores (see also
Langer et al., 2006; Langer et al., 2009).
Acknowledgements
We thank Friedel Hinz, Christiane Lorenzen and KarinWoudsma for laboratory assistance. G.L. acknowledges
financial support by the Spanish Ministry of Education(Juan de la Cierva programme) cofunded by the European Social Fund and Ministry of Science and Innovation.
This work was supported by the Spanish Ministry of Science and Innovation co-funded by the European Social
Fund (CTM2007-28909-E/MAR and CTM2008-04365-E)
and the ESF MERF project (ESF EuroCLIMATE ERASCT-2003-980490 of the European Commission, DG Research, Fp6.) Netherlands Organisatie Voor Wetenschappelijk Onderzoek (NWO 855.01.086), ANR BOOM
project (ANR-05-BIODIV-004), and EU FP7 ASSEMBLE
project (RI-227799). This research was supported by theEuropean Commission through grant 211384 (EU FP7"EPOCA"), and the German Federal Ministry of Education and Research (BMBF, FKZ 03F0608, "BIOACID").
The research leading to these results has received funding
from the European Community's Seventh Framework programme under grant agreement 265103 (Project MedSeA).
References
Bollmann, J. & Herrle, J.O. 2007. Morphological variationof Emiliania huxleyi and sea surface salinity. Earth andPlanetary Science Letters, 255(3-4): 273-288.
Borman, A.H., De Jong, E.W., Huizinga, M., Kok, D.J.,
Westbroek, P. & Bosch, L. 1982. The role in CaCO3
crystallization of an acid Ca2+-binding polysaccharid
associated with coccoliths of Emiliania huxleyi. European Journal of Biochemistry, 129: 179-183.
Feng, Y., Warner, M.E., Zhang, Y., Sun, J., Fu, F.-X.,
Rose, J.M. & Hutchins, D.A. 2008. Interactive effects
of increased pCO2, temperature, and irradiance on themarine coccolithophore Emiliania huxleyi (Prymnesiophyceae). European Journal of Phycology, 43: 87-98.
Fichtinger-Schepman, A.M.J., Kamerling, J.P., Versluis,
C. & Vligenhart, J.F.G. 1981. Structural studies ofthe methylated, acidic polysaccharide associated withcoccoliths of Emiliania huxleyi (Lohmann) Kamptner.
Carbohydrate Research, 93: 105-123.
Green, J.C., Heimdal, B.R., Paasche, E. & Moate, R.
1998. Changes in calcification and the dimensions ofcoccoliths of Emiliania huxleyi (Haptophyta) grown at
reduced salinities. Phycologia, 37: 121-131.
Guillard, R.R.L. & Ryther, J.H. 1962. Studies of marine

planktonic diatoms, I, Cyclotella nanna (Hustedt) and
Detonula convervacea (Cleve). Canadian Journal of
Microbiology, 8: 229-239.
Henriksen, K. & Stipp, S.L.S. 2009. Controlling Biomineralization: The Effect of Solution Composition on
Coccolith Polysaccharide Functionality. Crystal
Growth & Design, 9(5): 2088-2097.
Henriksen, K., Stipp, S.L.S., Young, J. & Marsh, M.E.
2004. Biological control on calcite crystallization:
AFM investigation of coccolith polysaccharide function. American Mineralogist, 89: 1586-1596.
Henriksen, K., Stipp, S.L.S., Young, J.R. & Bown, P.R.
2003. Tailoring calcite: Nanoscale AFM of coccolith
biocrystals. American Mineralogist, 88: 2040-2044.
Langer, G. & Benner, I. 2009. Effect of elevated nitrateconcentration on calcification in Emiliania huxleyi.
Journal of Nannoplankton Research, 30: 77-80.
Langer, G., De Nooijer, L.J. & Oetjen, K. 2010. On therole of the cytoskeleton in coccolith morphogenesis:
the effect of cytoskeleton inhibitors. Journal of Phycology, 46: 1252-1256.
Langer, G., Geisen, M., Baumann, K.-H., Kläs, J., Riebesell, U., Thoms, S. & Young, J.R. 2006. Species-specific responses of calcifying algae to changing seawater carbonate chemistry. Geochem. Geophys. Geosyst.,
7: Q09006, doi:09010.01029/02005GC001227.
Langer, G., Nehrke, G., Probert, I., Ly, J. & Ziveri, P.
2009. Strain-specific responses of Emiliania huxleyi
to changing seawater carbonate chemistry. Biogeosciences J1 - BG, 6(11): 2637-2646.
Marsh, M.E., Ridall, A.L., Azadi, O. & Duke, P.J. 2002.
Galacturonomannan and Golgi-derived membrane
linked to growth and shaping of biogenic calcite. Journal of Structural Biology, 139: 39-45.
Riebesell, U., Zondervan, I., Rost, B., Tortell, P.D., Zeebe,
R.E. & Morel, F.M.M. 2000. Reduced calcification ofmarine plankton in response to increased atmosphericCO2. Nature, 407: 364-367.
Royal Society, R. 2005. Ocean acidification due toincreasing atmospheric carbon dioxide, Policy Document 12/05, The Royal Society, London.
Takahashi, T., Sutherland, S.C., Sweeney, C., Poisson,
A., Metzl, N., Tilbrook, B., Bates, N., Wanninkhof,
R., Feely, R.A. & Sabine, C. 2002. Global sea-air CO2
flux based on climatological surface ocean pCO2, andseasonal biological and temperature effects. Deep Sea
Research Part II: Topical Studies in Oceanography,49(9-10): 1601-1622.
Trimborn, S., Langer, G. & Rost, B. 2007. Effects ofvarying calcium concentrations and light intensities on
calcification and photosynthesis in Emiliania huxleyi.
Limnology and Oceanography, 52: 2285-2293.
Watabe, N. & Wilbur, K.M. 1966. Effects of temperatureon growth, calcification, and coccolith form in Coccolithus huxleyi (Coccolithineae). Limnology andOceanography, 11: 567-575.
Young, J.R. 1994. Functions of coccoliths. In: A. Winter& W. G. Siesser (Eds.), Coccolithophores. Cambridge
University Press: 63-82.